Cartilage-derived Retinoic Acid-sensitive Protein (CD-RAP) is a newly discovered low molecular weight secretion protein synthesized exclusively by chondrocytes under normal conditions (Dietz and Sandell, 1996). This molecule caught and held our attention for the following reasons: First, it is co-regulated with type II collagen by retinoic acid and expressed only in cartilage in bovine and mouse embryos and adult rat. Second, CD-RAP has been isolated from melanoma cell lines and tumors as Melanoma Inhibitory Protein (MIA). The common function of the CD-RAP/MIA in these two tissues is unknown. During mouse development, CD-RAP expression in initiated at the beginning of chondrogenesis. In melanoma, CD-RAP/MIA is expressed early in tumor development, but not by normal melanocytes. The physiological function of CD-RAP/MIA in cartilage or melanoma may be related to the ability of CD-RAP/MIA to reduce DNA synthesis and cause rounding of cells in culture. To further investigate the function and regulation of CD-RAP, we have cloned and characterized bovine CD-RAP, and cloned and characterized the mouse gene. The overall hypothesis directing the present studies is: CD-RAP plays a role in establishing and maintaining the chondrocyte phenotype. The Specific Aims of the proposal are: (1) Determine the effect of removal of CD-RAP from the mouse genome by targeted disruption of the gene. (2) Determine the effect of overexpression of CD-RAP in mice using specific promoters to drive expression in the normal tissue, chondrocytes, and in a novel tissue, bone. (3) Investigate the regulation of CD-RAP in chondrocytes in vitro to lay the groundwork for control of pathological CD-RAP expression. (4) Investigate tissue specific regulation of CD-RAP gene expression in transgenic mice. Reagents and methods are currently in place to address the questions posed in this series of studies. In short, this is a new molecule that is chondrocyte-specific, small and secreted. As it is exquisitely specific to cartilage under normal conditions, it is a good candidate for an easily detectable marker for cartilage metabolism.